APOE Protects Against Severe Infection with Mycobacterium tuberculosis by Restraining Production of Neutrophil Extracellular Traps (scRNA-Seq)

Mice lacking apolipoprotein E (APOE, Apoe-/- mice) are highly susceptible to infection with Mycobacterium tuberculosis (Mtb) but the underlying immune dysregulation has been unclear. We found that Mtb infected Apoe-/- mice have significantly elevated levels of neutrophils in their lungs compared to controls and demonstrated that depleting neutrophils, depleting pDCs, blocking type1 interferon signaling, and blocking LTB4 receptor signaling all improved the outcome of Mtb-infected Apoe-/- mice. Neutrophils can undergo a specialized form of cell death termed NETosis in which activation of the enzyme PAD4 leads to extrusion of neutrophil extracellular traps (NETs). NETs have been associated with severe tuberculosis in other mouse models and in humans but a role of NETs in the pathology has not been directly established. We demonstrate that blocking PAD4 activation leads to a decrease in NETs in the lungs and, strikingly, completely reverses the hypersusceptiblity of Apoe-/- mice. To examine the role of Apoe in the responses of pulmonary immune cells to infection with Mtb in vivo, we fed B6, Apoe-/-, and Ldlr-/- mice a normal or high cholesterol diet, infected them with ~50 CFU of Mtb H37Rv via aerosol, and isolated CD45+, IV- cells from the lung prior to infection (day 0) or at 14 days following infection. This population was analyzed by single-cell RNA-seq using the 10X Genomics platform.

载脂蛋白E（apolipoprotein E, APOE）基因敲除小鼠（Apoe-/- mice）对结核分枝杆菌（Mycobacterium tuberculosis, Mtb）感染具有极高易感性，但其潜在的免疫失调机制尚不明确。本研究发现，与对照组相比，结核分枝杆菌感染的Apoe-/-小鼠肺部中性粒细胞水平显著升高；并证实，中性粒细胞耗竭、浆细胞样树突状细胞（pDCs）耗竭、阻断I型干扰素信号通路以及阻断白三烯B4（LTB4）受体信号通路，均可改善结核分枝杆菌感染的Apoe-/-小鼠的感染结局。
中性粒细胞可发生一种名为中性粒细胞胞外陷阱释放（NETosis）的特异性细胞死亡形式，此过程中肽酰精氨酸脱亚胺酶4（PAD4）的激活会介导中性粒细胞胞外陷阱（NETs）的释放。既往研究已在其他小鼠模型及人类样本中证实NETs与重症结核存在关联，但NETs在结核病理损伤中的作用尚未得到直接验证。本研究证实，抑制PAD4激活可降低小鼠肺部NETs水平，尤为值得注意的是，其可完全逆转Apoe-/-小鼠的结核易感表型。
为探究载脂蛋白E在肺部免疫细胞对结核分枝杆菌感染的体内应答中的作用，我们将C57BL/6（B6）、Apoe-/-及低密度脂蛋白受体基因敲除小鼠（Ldlr-/- mice）分别饲喂正常或高胆固醇饮食，通过气溶胶途径感染约50个菌落形成单位（CFU）的结核分枝杆菌H37Rv株；分别于感染前（第0天）或感染后14天，从小鼠肺部分离CD45阳性、活细胞（CD45+, IV- cells），并通过10X Genomics平台对该细胞群进行单细胞RNA测序。